PROJECTSUMMARY Retinitispigmentosa(RP)causesirreversibleblindnessinindividualsofallages.Itaffects~1in3,500people worldwide.WhileRPisinitiallycharacterizedbynightblindnessandperipheralvisionloss,mostRPpatients losetheircentralvisionandbecomelegallyblindbytheageof40.Thereisnotreatmenttosloworstop visionloss.Mislocalizationofthelight-absorbingproteinrhodopsinintherodsisacommonhallmarkshared bymanyanimalmodelsofRP.Thelong-termobjectiveofthisapplicationistodissectthemolecularpathway thatunderliesthesortinganddeliveryoftherhodopsinanddetermineitsdiseaserelevance.Thisinformation willacceleratethediscoveryofnewtreatmentsforRPandotherretinaldegenerativediseases.Inthe mammalianrods,whicharehighlycompartmentalized,rhodopsinissynthesizedinthebiosynthetic organellesconfinedtotheinnersegment.Rhodopsinisthenvectoriallydeliveredtoandconcentratedinthe outersegment(OS).Whilerhodopsin?straffickingthroughtheendoplasmicreticulum-Golgipathwayhas beeninvestigated,theimportanceoftheendosomeinrhodopsin?sOStargetingisunclear.Inmanyothercell types,theendosomes ervesasakeysortingstationforproteinsatthecrossroadsofmultipleintracellular traffickingpathways.Prolongedendosomalaccumulationofflyrhodopsinhasbeenshowntoleadtolight- dependentretinaldegeneration.Ourpreliminaryresultsshowedthatthenewly-synthesizedrhodopsin transitsthroughtheendosomalcompartmentsinmouserodsinvivo.TheOStargetingsignalofthe rhodopsinbindstoanearlyendosome-specificprotein,SARA.SARAdeficiencyinmouserodsnotonly causesrhodopsinmislocalizationbutalsoseveralothercellulardefectsintheendolysosomalsystem.Inthis application,wewilltestthecentralhypothesisthatinmammalianrodsthetrans-endosomalpathwaycritically regulatesthefidelityandtheefficiencyoftheOStargetingofrhodopsin.First,wewilladdresswhetherthe RPmutantrhodopsinsareretainedintheendosomesabnormallyduringtheirtransittotheOS,andwhether thisdefectperturbsthehomeostasisofotherendomembranes(Aim1).Wewillgeneratemultiple, complementarymousemodelstoexaminetheroleofthetrans-endosomalpathwayinthemorphogenesisof theOSanditsrhodopsinexpression(Aim2).Wewillalsoprofiletherodproteinsthattransitthroughthe endosomalsystemandcharacterizetheirinteractionwithkeyendosomaltraffickingregulators(Aim3).We willachievetheseaimsbyapplyingstate-of-the-arttechniquessuchasrod-specificinduciblegene expressionandgenedeletion,super-resolutionconfocalmicroscopy,correlativelight-electronmicroscopy, and3Dscanningelectronmicroscopy.Byprovidingmechanisticinsightsonphotoreceptorproteintrafficking andOSbiogenesis,thisresearchwillcontributetothedevelopmentofnewtherapiesforRPandrelated diseases.